In order to prevent epidemic/pandemic influenza, high growth reassortants (genetically mixed influenza viruses) are necessary for efficient preparation of inactivated influenza virus vaccines. Many viruses selected for use in vaccines do not grow well in eggs or in cell cultures in their native states. Generating high-growth influenza viruses is thus a priority for increasing vaccine production. In addition to the inactivated influenza virus vaccine, live attenuated influenza viruses stimulate not only the local and systemic antibody responses but also to have effects on cellular immune reactions, which could result in additional protection against influenza infection. Since replication is one of the potential factors in influenza virus virulence, understanding viral replication is a priority for preparing safe live attenuated vaccines. Thus, a better understanding of the mechanisms of replication of influenza viruses is central to improving both inactivated and live attenuated influenza vaccines. The matrix protein (M1) of influenza virus plays a central role in viral replication. Although multiple genes may contribute to the attenuation phenotype of influenza viruses, the matrix gene by itself may confer attenuating properties. We have studied the RNP-binding activity of M1s from a high-growth strain and a relatively low-growth wild-type strain. Our results indicated that RNP-binding of M1 of the high-growth strain was more difficult to break than the RNP-binding of M1 of the low-growth strain. These studies also suggested that the binding between M1 and RNP might be one of the factors determining viral growth (Liu and Ye, Virology, 2002). Another of our studies also demonstrated that the interaction of NP with viral RNA and M1 is necessary for maturation of the viral RNP and for final assembly of viral particle (Huang et al., Virology, 2001). Our current studies focus on introducing mutations into functional domains of wild type influenza virus M1 by reverse genetics to alter the protein binding capacities of M1. Viral replication and attenuation of the M1 mutants are being evaluated in cell cultures and in animals. Live attenuated influenza vaccines are licensed in US for the prevention of disease caused by influenza A and B viruses in the population of 5-49 years of age, but not for children under 5 years of age. Live attenuated influenza vaccines are reassortant (genetically mixed) viruses from attenuated donor strains and newly isolated wild-type (WT) influenza viruses. Although multiple genes may contribute to the attenuation phenotype of influenza viruses, the matrix (M) gene by itself confers part of the overall attenuation properties. However, a natural occurring, temperature sensitive virus with a single-site mutation on M gene is not genetically stable; reversion to virulent wild type virus can happen when the virus replicates in the host. The matrix protein (M1) of influenza virus plays a central role in viral replication. We have studied the RNP-binding activity of M1s from a high-growth strain and a relatively low-growth wild-type strain. Our results indicated that RNP-binding of M1 of the high-growth strain was more difficult to break than the RNP-binding of M1 of the low-growth strain. These studies suggested that the binding between M1 and RNP might be one of the factors determining viral growth. Another of our studies also demonstrated that the interaction of NP with viral RNA and M1 is necessary for maturation of the viral RNP and for final assembly of viral particle. Our current studies focus on introducing mutations into M1 functional domains of wild type influenza virus by reverse genetics to alter the protein binding capacities of M1. PROGRESS. Our recent in-vitro studies show that some single mutations (K102S or K104S) are lethal, but others (K101S or K105S) result in reduced viral replication at non-permissive temperature. Multiple mutations (SKLKS) result in temperature-sensitive phenotype. Attenuation phenotypes of M gene mutants are being evaluated in small animals.